Chromatographic media normally used for chromatofocusing of biomolecules are substituted with a suitable buffering and binding ligands homogeneous substituted in the beads to accomplish the pH-gradient and the separation. Chromatofocusing (CF) combines the advantage of ion-exchange procedures with the high resolution of isoelectric focusing into a single simple to use “isocratic” chromatographic focusing procedure. During chromatofocusing, a weak ion-exchange column of suitable buffering capacity is equilibrated with a buffer that defines the upper pH (in the case of anion exchange CF media) of the separation pH gradient to follow. A second “focusing” buffer is then applied to elute bound proteins, roughly in the order of their isoelectric (PI) points. The pH of the focusing buffer is adjusted to a pH that defines the lower limit of the pH gradient. The pH gradient is formed inside the packed column during isocratic elution with a single focusing buffer; no external gradient forming device is required. The pH gradient is formed as the eluting buffer (i.e., focusing buffer) titrates the buffering weak ion exchange groups on the ion exchanger. Peak widths in the range of 0.02-0.05 pH units and samples containing several hundred milligrams of protein can be processed in a single step. Focusing of the protein band occurs because the velocity of the mobile phase is higher than the velocity at which the generated pH gradient is developed and moves through the column. Faster transport of analytes down the column to a region with a pH that promotes binding than the movement of the binding region it self.
Chromatofocusing is a powerful analytical tool for characterization of amphoteric substances, such as proteins and large protein aggregates as e.g. virus, as well as an effective preparative technique for high purity protein isolation. The beads used in chromatofocusing are normally weak anion exchangers or ion exchangers with both strong and weak IEX ligands (e.g. PBE94, Mono P and DEAE media from GE Healthcare Biosciences AB) where the different amine ligands participate both in the separation process and the generation of the pH gradient through out the whole beads. Even with its prominence in protein separation and purification technology, the technology has remained mostly unchanged from the pioneering work of Sluyterman et al (J. Chromatography 150 (1978) 17-44).
New chromatographic polishing media for chromatofocusing (CF) with significantly improved loading capacities compared to existing CF media and improved peak shapes should be welcomed in the chromatographic area.